Nanosecond time-resolved and steady-state fluorescence techniques will be used to investigate the static and dynamic structures of proteins and macromolecular assemblies. Both intrinsic fluorophores and extrinsic fluorescence probes will be utilized. Procedures will be devised to differentiate complex decay having its origin in ground-state microheterogeneity from that which has its origin in excited-state interactions. The work will emphasize the use of "overdeterminations" with the development of analysis algorithms which combine the analysis of fluorescence decay data obtained at different wavelengths, temperature, pH and at various quencher concentrations. Efforts will be focused on the fluorescence of tryptophan and extrinsic probes such as substituted naphthalene derivatives and other fluorophores of interest. Fluorescence spectroscopy will be used to study the interactions of Enzyme I, HPr and III/Glc, three proteins of the phosphoenolpyruvate:glycose phosphotransferase system. Intrinsic fluorescence and resonance energy transfer, as measured by nanosecond fluorometry will be used to study the folding of Staphylococcal nuclease. The energy transfer donor will be the single tryptophan residue and the acceptor will be conjugated to single cysteine residues introduced at unique sites in the protein. Fluorescence studies will be carried out on the structure of horse liver alcohol dehydrogenase and thioredoxin. Excited-state solvent relaxation will be investigated in order to better understand how fluorescent probes sense changes in their environment. The focus of the research is to develop new ways to use steady-state and nanosecond time-resolved fluorescence to uncover points of ignorance regarding biological macromolecules and macro-assemblies.